Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Hantaan virus, the prototype virus of hemorrhagic fever with renal syndrome, was examined for nucleic acid characteristics which would support its previously proposed inclusion in the virus family Bunyaviridae. Nucleocapsid RNA from Hantaan virions and a control bunyavirus were examined for ribonuclease A (RNase A) sensitivity. Both viruses exhibited a similar accessibility of RNA within nucleocapsids to digestion by RNase A. Complete digestion of the RNA of both viruses was affected with high concentrations of ribonuclease. Evidence for negative strand RNA polarity was obtained by an in vitro transcriptase assay. RNA dependent RNA polymerase activity was associated with Hantaan virions. Polymerase activity required manganese and nucleoside triphosphates and was enhanced by magnesium, 2-mercaptoethanol, and sodium chloride. Oligonucleotide map analysis of the large (L), medium (M), and small (S) genome segments of Hantaan virus demonstrated that each RNA species was unique with respect to each other and was different from host cell ribosomal RNA. A common 3' terminal sequence of the three genome segments was determined to be 3' AUCAUCAUCUG. This sequence is different from those reported for viruses within the four recognized genera of the Bunyaviridae. Because all other data were consistent with nucleic acid characteristics of the Bunyaviridae, we propose a separate genus within the Bunyaviridae with Hantaan as its prototype virs.
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PMID:Analysis of Hantaan virus RNA: evidence for a new genus of bunyaviridae. 641 60

The genome of Lelystad virus (LV), a positive-strand RNA virus, is 15 kb in length and contains 8 open reading frames (ORFs) that encode putative viral proteins. ORFs 2 to 7 were cloned in plasmids downstream of the Sp6 RNA polymerase promoter, and the translation of transcripts generated in vitro yielded proteins that could be immunoprecipitated with porcine anti-LV serum. Synthetic polypeptides of 15 to 17 amino acids were selected from the amino acid sequences of ORFs 2 to 7 and antipeptide sera were raised in rabbits. Antisera that immunoprecipitated the in vitro translation products of ORFs 2 to 5 and 7 were obtained. Sera containing antibodies directed against peptides from ORFs 3 to 7 reacted positively with LV-infected alveolar lung macrophages in the immunoperoxidase monolayer assay. Using these antipeptide sera and porcine anti-LV serum, we identified three structural proteins and assigned their corresponding genes. Virions were found to contain a nucleocapsid protein of 15 kDa (N), an unglycosylated membrane protein of 18 kDa (M), and a glycosylated membrane protein of 25 kDa (E). The N protein is encoded by ORF7, the M protein is encoded by ORF6, and the E protein is encoded by ORF5. The E protein in virus particles contains one or two N-glycans that are resistant to endo-beta-N-acetyl-D-glucosaminidase H. This finding indicates that the high-mannose glycans are processed into complex glycans in the Golgi compartment. The protein composition of the LV virions further confirms that LV is evolutionarily related to equine arteritis virus, simian hemorrhagic fever virus, and lactate dehydrogenase-elevating virus.
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PMID:Characterization of proteins encoded by ORFs 2 to 7 of Lelystad virus. 783 70

Ebola (EBO) viruses were detected in specimens obtained during the hemorrhagic fever outbreak among humans in Kikwit, Democratic Republic of the Congo (DRC), in 1995 (subtype Zaire) and during an outbreak of disease in cynomolgus macaques in Alice, Texas, and the Philippines in 1996 (subtype Reston). Reverse transcriptase-polymerase chain reaction assays were developed and proven effective for detecting viral RNA in body fluids and tissues of infected individuals. Little change was seen in the nucleotide or deduced amino acid sequences of the glycoprotein (GP) of these EBO virus subtypes compared with those of their original representatives (i.e., the 1976 Yambuku, DRC, EBO isolate [subtype Zaire] and the 1989 Philippines and Reston, Virginia, isolates [subtype Reston]). The nonstructural secreted GP (SGP), the primary product of the GP gene, was more highly conserved than the structural GP, indicating different functional roles or evolutionary constraints for these proteins. Significant amounts of SGP were detected in acutely infected humans.
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PMID:Detection and molecular characterization of Ebola viruses causing disease in human and nonhuman primates. 998 80

The understanding of dengue virus pathogenesis has been hampered by the lack of in vitro and in vivo models of disease. The study of viral factors involved in the production of severe dengue, dengue hemorrhagic fever (DHF), versus the more common dengue fever (DF), have been limited to indirect clinical and epidemiologic associations. In an effort to identify viral determinants of DHF, we have developed a method for comparing dengue type 2 genomes (reverse transcriptase PCR in six fragments) directly from patient plasma. Samples for comparison were selected from two previously described dengue type 2 genotypes which had been shown to be the cause of DF or DHF. When full genome sequences of 11 dengue viruses were analyzed, several structural differences were seen consistently between those associated with DF only and those with the potential to cause DHF: a total of six encoded amino acid charge differences were seen in the prM, E, NS4b, and NS5 genes, while sequence differences observed within the 5' nontranslated region (NTR) and 3' NTR were predicted to change RNA secondary structures. We hypothesize that the primary determinants of DHF reside in (i) amino acid 390 of the E protein, which purportedly alters virion binding to host cells; (ii) in the downstream loop (nucleotides 68 to 80) of the 5' NTR, which may be involved in translation initiation; and (iii) in the upstream 300 nucleotides of the 3' NTR, which may regulate viral replication via the formation of replicative intermediates. The significance of four amino acid differences in the nonstructural proteins NS4b and NS5, a presumed transport protein and the viral RNA polymerase, respectively, remains unknown. This new approach to the study of dengue virus genome differences should better reflect the true composition of viral RNA populations in the natural host and permit their association with pathogenesis.
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PMID:Dengue virus structural differences that correlate with pathogenesis. 1023 34

Puumala (PUU) virus causes a form of hemorrhagic fever with renal syndrome (HFRS), called nephropathia epidemica (NE), in Europe. HFRS is characterized by an increased capillary permeability, which we hypothesize is caused by hyperactivation of the host immune system, especially cellular immune responses. To identify cytotoxic T lymphocytes (CTLs) specific for the PUU virus from NE patients, we have made recombinant vaccinia viruses expressing PUU virus proteins, the nucleocapsid (N) and two surface glycoproteins, G1 and G2. Recombinant vaccinia viruses carrying the N or the first half of the G2 cDNA under the control of a strong synthetic promoter were made. To express G1 and the second half of the G2 proteins, however, we needed to use a T7 expression system, where the T7 RNA polymerase is produced from another recombinant vaccinia virus co-infecting the same cells. These recombinant vaccinia viruses were used to detect and clone PUU virus-specific CTLs from the peripheral blood mononuclear cells of NE patients. An HLA-A24-restricted CTL line recognizing the G2 protein was isolated and its 9-mer epitope was determined.
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PMID:Generation of recombinant vaccinia viruses expressing Puumala virus proteins and use in isolating cytotoxic T cells specific for Puumala virus. 1190 Aug 40

The importance of leptospirosis in Southeast Asia was assessed in conjunction with other studies supported by the U.S. Naval Medical Research Unit No. 2 (US NAMRU-2), Jakarta, Republic of Indonesia. These included studies of hospital-based, acute clinical jaundice in Indonesia, Lao PDR, and Socialist Republic of Vietnam; nonmalarial fever in Indonesia; and hemorrhagic fever in Cambodia. Background prevalence estimates of leptospiral infection were obtained by a cross-sectional, community-based study in Lao PDR. Laboratory testing methods involved serology, microscopic agglutination test, and reverse-transcriptase polymerase chain reaction. Suggestive evidence of recent leptospiral infections was detected in 17%, 13%, and 3% of patients selected on the basis of non-hepatitis A through E jaundice, nonmalarial fever, and hemorrhagic fever (in the absence of acute, dengue viral infections). Leptospiral IgG antibody, reflective of prior infections, was detected in 37% of human sera, collected in Lao PDR. The predominant leptospiral serogroups identified from cases with clinical jaundice were Hurstbridge, Bataviae, and Icterohaemorrhagiae tonkini LT 96 69. Among the nonmalarial febrile cases, Bataviae was the most frequently recognized serogroup. Pyrogenes and Hurstbridge were the principal serogroups among the hemorrhagic fever case subjects. These findings further attest to the relative importance of clinical leptospirosis in Southeast Asia. The wide spectrum of clinical signs and symptoms associated with probable, acute, leptospiral infections contributes to the potential of significant underreporting.
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PMID:The importance of leptospirosis in Southeast Asia. 1240 67

Lassa virus is endemic to West Africa and causes hemorrhagic fever in humans. To facilitate the functional analysis of this virus, a replicon system was developed based on Lassa virus strain AV. Genomic and antigenomic minigenomes (MG) were constructed consisting of the intergenic region of S RNA and a reporter gene (Renilla luciferase) in antisense orientation, flanked by the 5' and 3' untranslated regions of S RNA. MGs were expressed under the control of the T7 promoter. Nucleoprotein (NP), L protein, and Z protein were expressed from plasmids containing the T7 promoter and internal ribosomal entry site. Transfection of cells stably expressing T7 RNA polymerase (BSR T7/5) with MG in the form of DNA or RNA and plasmids for the expression of NP and L protein resulted in high levels of Renilla luciferase expression. The replicon system was optimized with respect to the ratio of the transfected constructs and by modifying the 5' end of the MG. Maximum activity was observed 24 to 36 h after transfection with a signal-to-noise ratio of 2 to 3 log units. Northern blot analysis provided evidence for replication and transcription of the MG. Z protein downregulated replicon activity close to background levels. Treatment with ribavirin and alpha interferon inhibited replicon activity, suggesting that both act on the level of RNA replication, transcription, or ribonucleoprotein assembly. In conclusion, this study describes the first replicon system for a highly pathogenic arenavirus. It is a tool for investigating the mechanisms of replication and transcription of Lassa virus and may facilitate the testing of antivirals outside a biosafety level 4 laboratory.
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PMID:Replicon system for Lassa virus. 1556 87

Rift Valley fever virus (RVFV) causes massive mosquito-borne epidemics among humans and decimates ruminants in which the mortality rate is about 1% and 10-30%, respectively. Morbidity in RVFV-infected humans is high largely due to the effects of hemorrhagic fever and encephalitis. This virus is native to sub-Saharan Africa; yet if this virus is introduced into the environment, virus transmission appears to occur whenever sheep and cattle are present with abundant mosquito populations. RVFV is a negative-strand RNA virus which belongs to the family Bunyaviridae, genus Phlebovirus, and contains tripartite-segmented genomes (S, M, and L). S-segment is the ambisense genome, where N and NSs genes are coded in an antiviral-sense and viral sense S-segment, respectively. The inhibition of host mRNA synthesis, which is induced by the binding of NSs protein to RNA polymerase II transcription factor TFIIH, is the primary reason for the host-protein shut-off in RVFV-infected cells. Development of a RVFV reverse genetics system, which has not been accomplished yet, is important for the study of viral replication mechanisms, host virus interaction, viral pathogenicity as well as vaccine evaluation and development.
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PMID:[Rift Valley fever virus]. 1574 61

In general, Ebola viruses are well known for their ability to cause severe hemorrhagic fever in both human and nonhuman primates. However, despite substantial sequence homology to other members of the family Filoviridae, Reston ebolavirus displays reduced pathogenicity for nonhuman primates and has never been demonstrated to cause clinical disease in humans, despite its ability to cause infection. In order to develop a tool to explore potential roles for transcription and replication in the reduced pathogenicity of Reston ebolavirus, we developed an RNA polymerase I (Pol I)-driven minigenome system. Here we demonstrate successful Reston ebolavirus minigenome rescue, including encapsidation, transcription, and replication, as well as the packaging of minigenome transcripts into progeny particles. The Pol I-driven Reston ebolavirus minigenome system provides a higher signal intensity with less background (higher signal-to-noise ratio) than a comparable T7-driven Reston ebolavirus minigenome system which was developed simultaneously. Successful Reston ebolavirus minigenome rescue was also achieved by the use of helper plasmids derived from the closely related Zaire ebolavirus or the more distantly related Lake Victoria marburgvirus. The use of heterologous helper plasmids in the Reston ebolavirus minigenome system yielded levels of reporter expression which far exceeded the level produced by the homologous helper plasmids. This comparison between minigenomes and helper plasmids from different filovirus species and genera indicates that inherent differences in the transcription and/or replication capacities of the ribonucleoprotein complexes of pathogenic and apathogenic filoviruses may exist, as these observations were confirmed in a Lake Victoria marburgvirus minigenome system.
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PMID:RNA polymerase I-driven minigenome system for Ebola viruses. 1576 42

The filoviruses Marburg virus and Ebola virus (EBOV) quickly outpace host immune responses and cause hemorrhagic fever, resulting in case fatality rates as high as 90% in humans and nearly 100% in nonhuman primates. The development of an effective therapeutic for EBOV is a daunting public health challenge and is hampered by a paucity of knowledge regarding filovirus pathogenesis. This report describes a successful strategy for interfering with EBOV infection using antisense phosphorodiamidate morpholino oligomers (PMOs). A combination of EBOV-specific PMOs targeting sequences of viral mRNAs for the viral proteins (VPs) VP24, VP35, and RNA polymerase L protected rodents in both pre- and post-exposure therapeutic regimens. In a prophylactic proof-of-principal trial, the PMOs also protected 75% of rhesus macaques from lethal EBOV infection. The work described here may contribute to development of designer, "druggable" countermeasures for filoviruses and other microbial pathogens.
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PMID:Gene-specific countermeasures against Ebola virus based on antisense phosphorodiamidate morpholino oligomers. 1641 82


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